Sirtuins
Sirtuins were first identified in yeast as silence information regulators (SIRs), from which the family derives its name (Rine, J and Herskowitz, I., 1987 Genetics 116:9-22). Sirtuins are class III histone deacetylases (HDACs) that consume one nicotinamide adenine dinucleotide (NAD+) for every acetyl group they remove from a protein substrate (Landry, J et al., 2000 PNAS 97(11):5807-5811). Sirtuins are phylogenetically conserved from bacteria to humans and regulate cell functions by deacetylating both histone and nonhistone targets. Sir2 in yeast (Sirt1 in mammals) is the founding member of the sirtuin gene family, and its deacetylase activity is required for chromatin silencing (Buck et al., 2004 J. Leukocyte Bio 75:939-950).
There are seven human homologs of Sirt1 (Sirt1-7). The distinct subcellular localizations of the sirtuins also contribute to their diverse functions (Gan, L and Mucke, L 2008 Neuron 58:10-14). Sirt1, Sirt6, and Sirt7 reside predominantly in the nucleus and have been implicated in genomic stability and cell proliferation. Sirt1 is the most studied among mammalian sirtuins. Sirt2, which resides mostly in the cytoplasm, is involved in mitosis and differentiation of oligodendrocytes, likely through deacetylation of tubulins. Sirt3, Sirt4, and Sirt5 are localized in mitochondria, they may play a role in energy metabolism and responses to oxidative stress.
Sirt1 and Sirt2 play important roles in aging and neurodegeneration (Gan, L and Mucke, L 2008 Neuron 58:10-14). Sir2/Sirt1 promotes replicative life-span extension in yeast (Kaeberlein, M et al., 1999 Genes and Development 13:2570-2580), C. elegans (Tissenbaum, H. A., and Guarente, L., 2001 Nature 410:227-230), and Drosophila (Rogina, B. and Helfand, S. L. 2004, PNAS 101(45):15998-16003). However, whether increased Sirt1 activity promotes longevity also in mammals fed a normal diet has not yet been reported. The most studied nongenetic strategy to extend life span is caloric restriction, which activates sirtuin pathways (Kenyon, C. 2001 Cell 105:165-168).
Abnormal accumulation of misfolded proteins appears to play a pivotal role in diverse neurodegenerative diseases. Relevant molecules include Aβ peptides and tau in Alzheimer disease (AD), α-synuclein in Parkinson's disease (PD), TDP-43 in frontotemporal dementia (FTD), and mutant huntingtin in Huntington's disease (HD) (Muchowski, P. J. and Wacker, J. L. 2005 Nature Reviews, Neuroscience 6:11-22). Recent evidence has shown that inhibition of Sir2 activity or down-regulations of Sir2/Sirt1 levels improve pathology in a Drosophila model of Huntington's disease (Pallos, J. et al., Hum Mol Genet 2008, 17:3767-3775). Inhibition of Sirt2 was shown to rescue α-synuclein toxicity and modified inclusion morphology in a cellular model of Parkinson's disease. Genetic inhibition of Sirt2 via small interfering RNA similarly rescued α-synuclein toxicity (Outeiro, T. F. et al., Science. 2007, 317:516-519). The role of down-regulation of sirtuin function in rescuing pathogenesis of neurodegenerative diseases has recently been linked to a mechanism that involves tau phosphorylation. Nicotinamide, a sirtuin inhibitor, restores cognition in AD transgenic mice by selective reduction of Thr231-phospho-tau (Green, K. N. et al., J Neurosci. 2008 28(45):11500-11510). This particular species of tau has been reported to interfere with microtubule polymerization (Sengupta et al., 1998 Archives of Biochem & Biophys 357(2):299-309; Cho, J. and Johnson, G. V. W. 2004 J. of Neurochem 88:349-358), and is a commonly used biomarker for AD found in CSF (Ewers et al., 2007 Neurology 69:2205-2212). Nicotinamide also dramatically increased acetylated alpha-tubulin, a primary substrate of Sirt2, and MAP2c, both of which are linked to increased microtubule stability. Resveratrol (a polyphenol found in red wines) and other structure-related compounds are Sirt1-pathway modulators (Granados-Soto, V. 2003 Drug News Perspect, 16: 299-307). Although initial research focused on the role of sirtuins in life span extension especially in lower organisms, more recent studies also show that Sir2/Sirt1 activity can impact a wide array of proteins implicated in cardiovascular and metabolic diseases (Pillarisetti, S. 2008 Recent Patents Cardiovasc Drug Discov 3:156-164) as well as neurodegenerative diseases.
The effects and regulation of sirtuins under physiological and pathological conditions appear to be extremely complex. While still under debate, an emerging hypothesis proposes that increasing Sir2/Sirt1 positively regulates replicative aging (in dividing cells), while negatively impacting chronological aging (in non-dividing cells), including neurodegenerative diseases (Fabrizio, P. et al., 2005, Cell. 123:655-67; Pallos, J. et al., Hum Mol Genet 2008, 17:3767-3775). Pharmacological agents that modulate activity and expression of sirtuins may help with defining the precise roles of sirtuins in cardiovascular and metabolic diseases as well as the production, assembly, and degradation of pathogenic proteins, elucidating the etiology of neurodegenerative diseases and opening up new avenues for therapeutic intervention.
Neurodegenerative Diseases and Neuroprotection
Alzheimer's disease is characterized by the deposition and accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein, Aβ or β/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890, 1984; Masters et al., Proc. Natl. Acad. Sci. USA 82:4245-4249, 1985; Husby et al., Bull. WHO 71:105-108, 1993). Aβ is derived by protease cleavage from larger precursor proteins termed β-amyloid precursor proteins (APPs) of which there are several alternatively spliced variants. The most abundant forms of the APPs include proteins consisting of 695, 751 and 770 amino acids (Tanzi et al., Nature 31:528-530, 1988).
The small Aβ peptide is a major component that makes up the amyloid deposits of “plaques” in the brains of patients with Alzheimer's disease. In addition, Alzheimer's disease is characterized by the presence of numerous neurofibrillary “tangles”, consisting of paired helical filaments which abnormally accumulate in the neuronal cytoplasm (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. USA 83:4913-4917, 1986; Kosik et al., Proc. Natl. Acad. Sci. USA 83:4044-4048, 1986; Lee et al., Science 251:675-678, 1991). The pathological hallmark of Alzheimer's disease is therefore the presence of “plaques” and “tangles”, with amyloid being deposited in the central core of the plaques. The other major type of lesion found in the Alzheimer's disease brain is the accumulation of amyloid in the walls of blood vessels, both within the brain parenchyma and in the walls of meningeal vessels that lie outside the brain. The amyloid deposits localized to the walls of blood vessels are referred to as cerebrovascular amyloid or congophilic angiopathy (Mandybur, J. Neuropath. Exp. Neurol. 45:79-90, 1986; Pardridge et al., J. Neurochem. 49:1394-1401, 1987)
For many years there has been an ongoing scientific debate as to the importance of “amyloid” in Alzheimer's disease, and whether the “plaques” and “tangles” characteristic of this disease were a cause or merely a consequence of the disease. Within the last few years, studies now indicate that amyloid is indeed a causative factor for Alzheimer's disease and should not be regarded as merely an innocent bystander. The Alzheimer's Aβ protein in cell culture has been shown to cause degeneration of nerve cells within short periods of time (Pike et al., Br. Res. 563:311-314, 1991; J. Neurochem. 64:253-265, 1995). Studies suggest that it is the fibrillar structure (consisting of a predominant β-pleated sheet secondary structure), characteristic of all amyloids, that is responsible for the neurotoxic effects. Aβ has also been found to be neurotoxic in slice cultures of hippocampus (Harrigan et al., Neurobiol. Aging 16:779-789, 1995) and induces nerve cell death in transgenic mice (Games et al., Nature 373:523-527, 1995; Hsiao et al., Science 274:99-102, 1996). Injection of the Alzheimer's Aβ into rat brain also causes memory impairment and neuronal dysfunction (Flood et al., Proc. Natl. Acad. Sci. USA 88:3363-3366, 1991; Br. Res. 663:271-276, 1994).
Parkinson's disease is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer, Berlin, pp. 920-933, 1912; Pollanen et al., J. Neuropath. Exp. Neurol. 52:183-191, 1993), the major components of which are filaments consisting of α-synuclein (Spillantini et al., Proc. Natl. Acad. Sci. USA 95:6469-6473, 1998; Arai et al., Neurosci. Lett. 259:83-86, 1999), an 140-amino acid protein (Ueda et al., Proc. Natl. Acad. Sci. USA 90:11282-11286, 1993). Two dominant mutations in α-synuclein causing familial early onset Parkinson's disease have been described suggesting that Lewy bodies contribute mechanistically to the degeneration of neurons in Parkinson's disease and related disorders (Polymeropoulos et al., Science 276:2045-2047, 1997; Kruger et al., Nature Genet. 18:106-108, 1998).
In AD, inflammatory reactions in the supporting neuronal cell network, especially microglia and astrocytes, can contribute to neuronal cell death. Overexpression of Sirt1 was found to inhibit NF-κB and block the neurotoxicity of beta-amyloid accumulation, resulting in neuroprotection (Chen, J., et al 2005 J. Biol. Chem. 280(48):40364-40374). In another study, AD transgenic mice, subjected to caloric restriction were found to have elevated Sirt1 activity in brain tissue and the corresponding classical Aβ neuropathology was prevented. This same study also demonstrated that in CHO-APPswe cells, that Sirt1 expression promoted α-secretase activity and attenuated Aβ peptide generation (Qin, W., et al., 2006 J. Biol. Chem. 281(31):21745-21754). A more recent paper describes a study where mouse models for AD and amyotrophic lateral sclerosis (ALS) treated with Sirt1 activating molecules promoted neuronal survival (Kim, D., et al. 2007 EMBO J 26:3169-3179).
In contrast to these studies, Chong Z., et al 2005 Curr Neurovasc Res. 2(4):271-285 showed that the sirtuin inhibitor nicotinamide enhanced neuronal cell survival. Li, Y., et al., 2008 Cell Metab. 8(1):4-5 have also shown that Sirt1 inhibition is neuroprotective.
In a Parkinson's disease model system, Outeiro, T. F., et al., (2007 Science 317:516-519) reported that human neuroglioma cell cultures induced for α-synuclein mediated toxicity could be rescued by treatment with siRNA inhibitors of Sirt2. In a Drosophila Huntington's disease model, flies heterozygous for Sir2 (ortholog of Sirt1) null mutations exhibited improved survival (Pallos, J., et al 2008 Hum. Mol. Gen. 17(3):3767-3775).
The common thread which links these diseases is that either an accumulated misfolded protein or something else in the brain exhibits neurotoxic properties which contributes to the progression of the disease and manifestation of symptoms. Studies have shown that modulation of sirtuin1 exhibits neuroprotective effects in many different models of these neurodegenerative diseases. Therefore, compounds which modulate levels of sirtuin1 could be therapeutically beneficial in the management of neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease, including Parkinson's plus diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and dementia with Lewy bodies (DLB).